This invention relates to modified herpesviruses and to materials and methods for their preparation, and to their uses, including uses in assays, in diagnostics and in immunotherapy and immunoprophylaxis.
The transport properties of VP22 protein are described for example in PCT WO 97/05265 (O""Hare and Elliott). Also described in that document are fusion products of VP22, including a fusion protein of VP22 with green fluorescent protein.
The present invention aims to provide herpesviral preparations comprising modified VP22 proteins, and a number of uses of such preparations.
An aspect of the invention provides herpesviral particles, e.g. a preparation of herpesviral particles isolated from the cell culture in which such particles were produced, in which at least part of the VP22 tegument protein is present as a fusion polypeptide comprising a VP22-active sequence and a non-VP22 peptide or polypeptide sequence selected from (a) sequences providing a detectable gene product, e.g. as a fusion with a gene encoding green fluorescent protein (GFP), and (b) immunogenic sequences corresponding to antigens associated with human disease.
The invention further provides herpesviral particles, e.g. a preparation of herpesviral particles isolated from the cell culture in which such particles were produced, in which at least part of the VP22 tegument protein is present in the form of a recombinant mutant form of VP22, for example, in the form of a recombinant fusion polypeptide comprising a VP22-active sequence and a non-VP22 peptide or polypeptide sequence. Among the herpesviral particles that can be produced in accordance with this aspect of the invention are particles with or without viral genomes, for example infectious herpesvirus particles, killed herpesviral particles, light particles and amplicons, (all of which can be made by techniques readily adaptable from or in accordance with per-se-known technique for producing herpesviral particles from virus types that do not incorporate the features described herein).
Also provided by the invention are DNA preparations including for example viral genomic DNA preparations in which there is present a VP22 recombinant fusion gene encoding a recombinant VP22 fusion polypeptide as already mentioned. The fusion polypeptide can for example be one that is detectable by fluorescence, e.g. a fusion protein derived from VP2 and green fluorescent protein (GFP).
In certain examples, the whole of the VP22 component of the virion can be of such a recombinant protein. For example, in a modified herpesvirus according to an example of the invention, the native VP22-encoding gene can have been replaced by a hybrid gene encoding a fusion product of VP22 with GFP.
Alternatively the viral particles can comprise normal/native VP22 along with modified or heterologous VP22. Such an example of the invention can e.g. comprise a mutant HSV which is gH-, i.e. deletant in respect of the essential viral gene encoding gH glycoprotein, and which has had a gene encoding a fusion product of VP22 and GFP inserted, under control of a promoter such as CMV IE promoter, at the locus of the deleted essential gene.
The herpesviral particles can be infectious herpesvirus, or otherwise can be of killed herpesvirus, or otherwise inactivated herpesvirus, or can be of defective herpesviral particles (e.g. herpesviral amplicons).
An infectious herpesvirus with a modified VP22 comprised in its tegument can be an attenuated virus, e.g. a virus carrying an attenuating mutation in its genome. xe2x80x98Attenuating mutationxe2x80x99 in this context is understood to comprise (i) mutations that reduce the virulence of the virus without preventing its replication on normal host cells, e.g. a virus with a tk-mutation, as well as (ii) lethal mutations in the presence of which the virus can only be grown on host cells that complement the lethal mutation, e.g. a virus with a gH- mutation.
The herpesvirus can be a mutant in which the sole copy of the VP22 gene has been mutated to encode the modified VP22 protein. Alternatively it can be a mutant in which a modified or heterologous VP22 gene has been inserted, with or without deletion of the native VP22 gene.
Alternatively again, the herpesvirus can be of wild-type in respect of its VP22 gene and can be grown and produced by infection of a host cell that expresses a modified or heterologous VP22. Under these circumstances the herpesvirus can if desired have a wild-type genome either entirely or at least in respect of VP22, although its tegument carries modified VP22 protein.
The invention provides isolated preparations of such herpesviral particles, i.e. substantially separated from the cell culture and medium that produced them, including pharmaceutical forms of such herpesviral particles, e.g. suitable for injection into a subject to be treated therewith, or inoculation into a cell preparation to be treated therewith for later pharmaceutical use.
The virus particles can be based on herpes viruses of various species. For example they can be based on herpes simplex virus, HSV1 or HSV2, or on VZV, BHV, EHV or MDV, among others.
The modified viruses can be used in a variety of ways. For example, they can be used as vaccines or vaccine components to provoke immune responses against the peptide fused with the VP22. Suitable antigens for incorporation in thsi connection can for example be those listed in WO 96/26267 (Cantab Pharmaceuticals Research Ltd).
In the case of viruses where the fused VP22 polypeptide comprises a deteactable protein such as for example GFP protein, the modified viruses can be used wherever simple fluorescent identification or detection of virus particles is desired, for example to detect virus particle formation at low levels in infected cell preparations.
For example, the invention further provides a process of using herpesviral particles where the fusion polypeptide sequence comprises a sequence providing a detectable VP22 fusion protein, to detect the progress of herpesviral infection of cells; the process comprises (i) contacting said particles with said cells and (ii) detecting said fusion protein within said cells. This can be particularly convenient where the protein is a fluorescent fusion protein and the fluorescence of the fusion protein is detected within the cells, e.g. fluorescence of GFP-VP22 fusion protein. The process can be used to detect the progress of cell infection by virus and/or to screen for neutralising antibody or inhibitors of infection of cells by virus.
Thus, for example, in this aspect of the invention, a process for using infectious viral particles as described herein comprises for example a test method such as a screening method for detecting the neutralisation of herpesviral particles: the process can comprise (a) treating infectious viral particles with a possibly-neutralising condition that is to be the subject of the test, e.g. treating the virus to a possible neutralising agent under test, such as for example a serum sample possibly containing a neutralising antibody; wherein, in the virus particles so treated, a gene encoding VP22 is present as a fusion with a gene having a detectable gene product, e.g. as a fusion with a gene encoding GFP, (or wherein the genome has another gene inserted or modified therein so that such gene has a readily-detectable gene product not normally expressed by corresponding wild-type virus, e.g. GFP or a fusion protein including GFP); (b) using said treated virus particles to infect host cells, and examining said host cells for the production therein of said detectable gene product. Presence of neutralising conditions can be sensitively and easily detected e.g. simply by observing green fluorescence or its absence in the culture of infected cells in the case where the gene product is GFP or a fusion protein related thereto: the fluorescence observed is then inversely related to the extent of virus neutralisation by the possibly neutralising conditions under test, and complete neutralisation can often be seen easily by absence of development of fluorescence compared to the result with an appropriate parallel control process using infectious virus.
Correspondingly, the process can be simply modified to function as a screen for any condition that is possibly inhibitory of virus replication.
Especially in this aspect of the invention, any of a variety of detectable genes and gene products can be used instead of GFP if desired: for example betagalactosidase gene and gene product or luciferase gene and gene product, both known per-se. In the case of beta-galactosidase, the gene product can be visualised and/or quantitated in the infected host cells in per-se known manner by a suitable substrate reaction, and the luciferase gene product can also be detected or quantitated in the infected cells by a suitable per-se known photogenic substrate. The GFP example is especially advantageous because its fluorescence can be immediately and simply visualised and requires no extra processing steps such as.
Other peptides incorporated for this purpose can be for example antigenic polypeptides, such as antigens of herpesvirus or papillomavirus, or of bacterial antigens against which an immune response is desired. Such preparations can for example be formulated in any suitable way known per se for viral vaccines.